Treadmill Exercise Training Ameliorates Functional and Structural Age-Associated Kidney Changes in Male Albino Rats
Aging is a biological process that impacts multiple organs. Unfortunately, kidney aging affects the quality of life with high mortality rate. So, searching for innovative nonpharmacological modality improving age-associated kidney deterioration is important. This study aimed to throw more light on the beneficial effect of treadmill exercise on the aged kidney. Thirty male albino rats were divided into three groups: young (3-4 months old), sedentary aged (23-24 months old), and exercised aged (23-24 months old, practiced moderate-intensity treadmill exercise 5 days/week for 8 weeks). The results showed marked structural alterations in the aged kidney with concomitant impairment of kidney functions and increase in arterial blood pressure with no significant difference in kidney weight. Also, it revealed that treadmill exercise alleviated theses effects in exercised aged group with reduction of urea and cystatin C. Exercise training significantly decreased glomerulosclerosis index, tubular injury score, and % area of collagen deposition. Treadmill exercise exerted its beneficial role via a significant reduction of C-reactive protein and malondialdehyde and increase in total antioxidant capacity. In addition, exercise training significantly decreased desmin immunoreaction and increased aquaporin-3, vascular endothelial growth factor, and beclin-1 in the aged kidney. This study clarified that treadmill exercise exerted its effects via antioxidant and anti-inflammatory mechanisms, podocyte protection, improving aquaporin-3 and vascular endothelial growth factor expression, and inducing autophagy in the aged kidney. This work provided a new insight into the promising role of aerobic exercise to ameliorate age-associated kidney damage.
The aging process is inevitable and culminates in the functional deterioration of multiple systems . One of the most affected organs by the normal aging process is the kidney . Renal aging has attracted attention because elderly persons are more susceptible to acute kidney injury and chronic kidney disease [3, 4]. Chronic kidney disease is more common as people get older, rising from 4% in people under the age of 40 to 47% in those aged 70 or older . Age-related changes in the kidneys include both anatomical and physiological alterations . These changes impede the kidney’s recovery from injury . Even with healthy aging, the number of functioning nephrons declines progressively. Approximately 6200 nephrons are lost per year after the age of 30 .
The aged kidney undergoes various structural changes, including glomerulosclerosis, podocyte injury, tubular atrophy, and microvascular changes . Additionally, many aspects of kidney functions are affected, such as glomerular filtration rate, glomerular basement membrane permeability, urine dilution/concentration, acid-base balance, hormonal activity, sodium chloride homeostasis, and control of arterial blood pressure .
Aquaporins (AQPs) are a family of transmembrane channels that mainly transport water. Eight AQPs are expressed in the kidney to maintain normal urine concentration. AQP3 is a water-/glycerol-transporting channel that facilitates the transport of water, urea, and glycerol . It also regulates several intracellular signaling processes involved in cell proliferation and apoptosis via permeating glycerol and hydrogen peroxide transport .
According to clinical and experimental studies, oxidative stress and inflammation have a significant role in renal aging . Moreover, the aging pathogenesis is aided by the decline in the production of proangiogenic growth factors and cytokines in aged animals, including vascular endothelial growth factor (VEGF), fibroblast growth factor, and transforming growth factor-beta .
Autophagy impairment has been implicated in the pathogenesis of many diseases, the development of multiple organ dysfunction, and other negative sequelae . Recently, it was discovered that the dysregulation of autophagy is involved in the pathogenesis of renal aging . Autophagy is a process of cellular recycling involving self-degradation and renewal of protein aggregates and damaged organelles to maintain cellular homeostasis and cell integrity . Beclin-1 is autophagy-related protein involved in the autophagy flux, regulating autophagosome formation [18, 19].
Aerobic exercise has been established to have a renoprotective effect in different experimental models of nephropathy [20, 21]. Moreover, it improves kidney function in patients with chronic kidney disease and enhances renal recovery after acute kidney injury [22, 23]. However, due to limited research, the effect of exercise on healthy renal aging is not well understood.
Thus, this study investigates the effect of treadmill exercise on structural and functional kidney changes in aged albino rats with referral to some underlying mechanisms. To the best of our knowledge, this is the first study to examine the role of exercise on the modulation of desmin (a marker for podocyte injury), VEGF, AQP3, and beclin-1 immunoreactivity in the kidneys of aged albino rats.
2. Materials and Methods
The protocols for animal care and use in this study were approved by the Institution Research Ethics Committee (Institutional Review Board approval number: 11\2021PHYS3. All experimental procedures and animal handling were strictly performed according to the European Committee Directive 86/609/EEC for animal experiments. In this study, thirty male Sprague-Dawley albino rats were used. The animals were housed in standard conditions with a natural light-dark cycle. They were fed standard rat chow and allowed access to water ad libitum. For proper acclimatization, animals were left undisturbed for one week before the start of the experiment.
2.2. Experimental Design
Rats were divided into three groups (10 rats per group): (1) young rats (3-4 months old, weighing 150–180 g) were kept sedentary throughout the study period; (2) sedentary aged rats (aged-S: 23-24 months old, weighing 350–400 g) were kept sedentary throughout the study period; (3) exercised aged rats (aged-Ex: 23-24 months old, weighing 350–400 g) performed moderate-intensity treadmill exercise five days/week for eight weeks.
Twenty-four hours after the last exercise session, systolic blood pressure was measured using a rat tail sphygmomanometer (Harvard Apparatus, Ltd., Edenbridge, England) connected to a pneumatic transducer (Harvard Apparatus, UK). Blood pressure changes were recorded via a physiograph (MK–III–S, Narco Bio-Systems, USA). Then, 24-hour urine samples were collected to measure urinary flow and creatinine. After that, fasting retroorbital blood samples were collected, and plasma was separated and stored at −80°C until biochemical analysis was conducted. Finally, all rats were weighed and then were sacrificed by cervical dislocation under anesthesia with 40 mg/kg intraperitoneal phenobarbital injection. The kidneys were immediately dissected, weighed, and preserved in 10% neutral buffered formalin for further histological and immunohistochemical studies.
2.3. Exercise Training Protocol
A multilane motor-drive treadmill was used to exercise the rats in the aged-Ex group, five days/week for eight weeks. The treadmill was custom-made as constructed by Rubin and Mickle . Initially, rats were allowed to be familiar with the treadmill for three days before starting the training. On the first day of familiarization, the rats were placed on a static treadmill for 10 minutes. In the second and third days, they ran for 10 min/day at 10 meters (m)/min with no inclination . During the familiarization phase, the rats that did not run on the treadmill were eliminated and replaced with new ones. Then, they were trained for 10 min/day at 10 m/min with no inclination. Over one week, treadmill speed was increased gradually to 20 m/min, maintaining this speed till the end of the study. On the other hand, the duration of the exercise period was progressively increased by 5 min every two days until reaching 60 min/day, which was maintained till the end of the study. The 20 m/min protocol was determined as an aerobic level in rats, corresponding to 50%–70% maximum oxygen consumption, which effectively alleviated early diabetic nephropathy in rats . The sedentary rats stayed on a turned-off treadmill for the same period.
2.4. Relative Kidney Weight
The relative kidney weight was calculated using the mean of the right and left kidney weights, corrected for body weight (g/100 g body weight).
2.5. Urine Collection
The rats were placed in individual metabolic cages for 24-hour urine sample collection. After measuring urine volumes, urine samples were centrifuged at 1000 revolutions per minute (rpm) for 10 min to remove cells and debris; then, the supernatant was separated and stored at −20°C to determine the creatinine levels.
2.6. Blood Sampling
After overnight fasting for 12 hours, blood samples were collected under light anesthesia from the retroorbital venous plexus using heparinized capillary tubes. Then, plasma was separated by centrifugation at 3000 rpm for 15 min and stored at −20°C until biochemical analysis was conducted.
2.7. Biochemical Analysis
Creatinine concentrations in plasma and urine and urea concentrations in plasma were determined using colorimetric kits (Spectrum Diagnostics, Egypt), whereas plasma concentrations of cystatin C, interleukin-6 (IL-6), and C-reactive protein (CRP) were determined using the corresponding rat enzyme-linked immunosorbent assay kits (cystatin C: ab201281; IL-6: ab100772; CRP: ab108827, Abcam, Cambridge, UK) according to the manufacturer’s instructions. Plasma malondialdehyde (MDA) was determined by thiobarbituric acid-reactive substances and total antioxidant capacity (TAC), representing the cumulative effect of all antioxidants present in plasma were determined using colorimetric kits (Biodiagnostic Company, Dokki, Giza, Egypt) according to the manufacturer’s instructions.
2.8. Creatinine Clearance
Creatinine clearance was calculated as a ratio of urine creatinine concentration (mg/dL) multiplied by urine volume (mL/min) to plasma creatinine concentration (mg/dL), with values expressed in mL/min .
2.9. Histological Study
The kidneys were fixed in 10% buffered formalin for 24 h, dehydrated in ascending grades of alcohol (70%–100%), and cleared and embedded in paraffin. Five-micron thick sections were cut by a microtome and subjected to hematoxylin and eosin (H&E) staining for routine histological examination, periodic acid-Schiff (PAS) stain, and Mallory’s trichrome stain. Then, the renal cortex was examined under a light microscope.
2.10. Semiquantitative Assessment of Tubular Injury Scoring
Using H&E-stained renal sections, we examined at least 20 cortical fields (20 x) in each section for tubular injury scoring based on the percentage of affected tubules as follows: score 0, no tubular injury; score 1, <10% of tubules injured; score 2, 10%–25% of tubules injured; score 3, 26%–50% of tubules injured; score 4, 51%–75% of tubules injured; score 5, >75% of tubules injured at the cortex . The grading percentage was calculated as follows: injury score (%) = (number of injured tubules/number of whole tubules) x 100, which was performed by an observer blinded to the groups under study.
2.11. Semiquantitative Estimation of Glomerulosclerosis Index
Using PAS-stained renal sections, we utilized a semiquantitative score to evaluate the degree of glomerulosclerosis according to Schaier et al. . The severity of the lesions was examined in 30 randomly selected glomeruli, graded from 0 to 4+ points according to the percentage of glomerular tuft involvement, and scored as follows: 0, 0%; 1+, 1%–25%; 2+, 26%–50%; 3+, 51%–75%; 4+, 76%–100%. The glomerulosclerosis score was calculated as follows: [(1 × number of glomeruli with +1) + (2 × number of glomeruli with +2) + (3 × number of glomeruli with +3) + (4 × number of glomeruli with +4)]/total number of the examined glomeruli, which was performed by an observer blinded to the groups under study.
2.12. Immunohistochemical Study
Paraffin sections (5 μm thick) were deparaffinized in xylene for 1–2 min, rehydrated in descending grades of ethanol (100%, 95%, and 70% ethanol) using two changes 5 min each, and rinsed in tap water. To block endogenous peroxidase, they were embedded in 3% H2O2 for 10 min. Moreover, the sections were treated with 2% trypsin at 37°C for 10 min and then subjected to antigen retrieval. Phosphate-buffered saline (PBS) and 10% normal goat serum (a blocking solution) blocked nonspecific protein binding. The sections were incubated with primary anti-desmin antibody (mouse monoclonal antibody, Abcam, ab8470, dilution 1 : 100), anti-VEGF antibody (rabbit polyclonal antibody, Lab Vision, USA, RB-222-P, dilution 1 : 100), anti-AQP3 antibody (rabbit polyclonal antibody, Abcam, ab125045, dilution 1 : 500), and anti-beclin-1 antibody (rabbit polyclonal antibody, Abcam, ab217179, dilution 1 : 200). The slides were incubated with the diluted primary antibody using PBS for 30 min. After that, a biotinylated goat polyvalent secondary antibody was applied. Drops of streptavidin peroxidase were added to the slide; then, the sections were left for 20 min and washed with PBS for 5 min. Finally, the prepared DAB substrate chromogen (3,3′-diaminobenzidine tetrahydrochloride) was added to the slides. Subsequently, the slides were washed with distilled water. Finally, the sections were counterstained with Harris’s hematoxylin.
2.13. Quantitative Assessment
Using ImageJ software, version K 1.45, the percentage area of collagen deposition and percentage area of desmin, VEGF, AQP3, and beclin-1 immunoreactivity were measured. For each parameter, ten nonoverlapping fields (40 x) for every specimen were randomly taken using a Leica DML B2/11888111 microscope equipped with a Leica DFC450 camera.
2.14. Statistical Analysis
The SPSS version 23 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Continuous data were expressed as the mean ± standard deviation (SD), whereas the ordinal data (tubular scoring) were expressed as median and range. The significance of differences between groups in all examined parameters was determined by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test, whereas tubular injury scoring was determined by the Kruskal–Wallis test followed by post hoc Mann-Whitney U test. values < 0.05 were considered statistically significant.
3.1. Body Weight and Relative Kidney Weight
At the end of the study, aged-S rats showed significantly higher body weight values than young rats (). Treadmill training resulted in a significant reduction in body weight compared with that in aged-S rats (). An insignificant difference was observed in the relative kidney weight between the experimental groups () (Table 1).
3.2. Biochemical Results
Regarding markers of renal function, aged-S rats showed significantly higher plasma creatinine, urea, and cystatin C levels than young rats (, , and , resp.). Treadmill training resulted in a significant reduction in plasma urea and cystatin C levels in the aged-Ex group compared with that in aged-S rats ( and , resp.), whereas an insignificant difference was observed in plasma creatinine between aged-S and aged-Ex rats (). Moreover, compared with the younger rats, aged-S rats showed significantly lower creatinine clearance (). An insignificant difference was observed between aged-S and aged-Ex rats in terms of creatinine clearance () (Table 1).
Regarding oxidative stress markers, aged-S rats showed significantly higher plasma MDA and significantly lower total antioxidant capacity than young rats (). Treadmill training in the aged-Ex group resulted in a significant reduction in plasma MDA and a significant increase in plasma total antioxidant capacity compared with those in aged-S rats ( and , resp.) (Table 1).
Regarding inflammatory markers, aged-S rats showed significantly higher plasma IL-6 and CRP levels compared with those in young rats ( and , resp.). Treadmill training in the aged-Ex group resulted in a significant reduction in plasma CRP () and insignificant change in plasma IL-6 compared with aged-S rats () (Table 1).
3.3. Systolic Blood Pressure
Aged-S rats showed significantly higher systolic blood pressure than young rats (). Treadmill training in the aged-Ex group resulted in a significant reduction in systolic blood pressure values compared with the corresponding values in the aged-S rats () (Figure 1).
3.4. Histological Results
3.4.1. Hematoxylin and Eosin (H&E) Results
The renal cortex of young rats showed normal renal corpuscles and tubules (Figure 2). Aged-S rats had marked renal degenerative changes. Some renal corpuscles exhibited degenerated glomerular tuft and widening of Bowman’s space, whereas others showed absence or narrowing of Bowman’s space. Most of the renal tubules had disturbed architecture, displaying exfoliation of their lining epithelium, pyknotic nuclei, vacuolated cytoplasm, and marked dilatation. The interstitial areas showed marked interstitial hemorrhage and intense inflammatory infiltrate. Moreover, thickened blood vessels with perivascular inflammatory infiltrates were observed (Figure 3).
On the contrary, treadmill training in the aged-Ex group alleviated most of the age-related damaging effects in the kidney. Slight exfoliation, mild interstitial hemorrhage, and some pyknotic nuclei were still reported in some tubules. In addition, few glomeruli showed congested capillaries (Figure 4).
3.5. Semiquantitative Assessment of Tubular Injury Scoring
A significant increase was observed () in tubular injury score in the aged-S group compared with that in the young ones. Moreover, compared with the aged-S group, the aged-Ex group had a significant decrease () in tubular injury score (Figure 5(a)).
3.5.1. Periodic Acid-Schiff (PAS) and Glomerulosclerosis Index (GSI)
Compared with the young group, PAS-stained renal sections of the aged-S rats showed deposition of PAS-positive material within the glomerulus tuft (sclerotic changes) with thickening of the glomerular basement membrane. Deposition of PAS-positive material, mostly cast formation, was also noted. These changes were ameliorated in the aged-Ex group (Figure 6).
The degree of glomerulosclerosis was significantly increased () in the GSI in the aged-S group compared with the young group. Compared with the aged-S group, the treadmill exercise significantly lowered GSI in the aged-Ex group () (Figure 5(b)).
3.6. Mallory’s Trichrome Results
Mallory’s trichrome-stained renal sections revealed intense fibrosis in the aged-S group that was decreased by exercise, indicated by the significant increase () in percentage area of collagen deposition in the aged-S renal sections compared with that in the young rats. In contrast, treadmill training significantly decreased collagen deposition in the aged-Ex group () compared with that in the aged-S group (Figure 7).
3.7. Immunohistochemical Results
The renal tissues of the aged-S group showed significantly higher () desmin immunoreactivity, indicating podocyte injury, than the young group. However, the aged-Ex group exhibited significantly decreased () desmin immunoreactivity compared with that of the aged-S group. A significant decrease was noted () in the VEGF, AQP3, and beclin-1 immunoreactivity within the renal tissue of the aged-S group compared with those of the control. On the contrary, treadmill exercise significantly increased () VEGF, AQP3, and beclin-1 immunoreactivity in the aged-Ex group compared with those in the aged-S group. Furthermore, regarding the different immunoreactions, a significant difference was observed () between the aged-Ex rats and the controls (Figures 8 and 9).
Recently, the aging kidney became a topic of great interest in geriatric medicine and clinical nephrology . This study states that treadmill exercise training may play a pivotal role in mitigating kidney aging. Moreover, to the best of our knowledge, this is the first study focusing on different mechanisms by which exercise affects the changes in the aged kidney.
Consistent with a previous report , aged-S rats had impaired renal function, reflecting the structural tubular changes and impairment of the glomerular filtration rate as postulated by Musso et al. . This was in line with the results in this study, confirmed by the significant increase in tubular injury score in the aged-S group compared with that of the young group.
On the contrary, exercise training improved plasma urea and cystatin C levels; however, it had an insignificant effect on creatinine and creatinine clearance. Dharnidharka et al.  clarified that serum cystatin C is a better filtration marker than serum creatinine, which could be due to the influence of physical exercise and muscle mass on plasma and urinary creatinine but not cystatin C levels . One potential explanation for the insignificant difference in creatinine and creatinine clearance between the aged-S and aged-Ex groups may be the probability of increased muscle mass in the exercised group and the worse kidney function in the sedentary rats. The significant reduction in body weight of aged-Ex rats compared with aged-S rats could be attributed to the improved renal function with exercise training and subsequently decreased fluid retention .
In this research, the impaired renal function in aged-S rats was consistent with histopathological alterations. The renal cortex was chosen for histopathological assessment because it is more affected by age than the medulla . The reported histopathological changes in this study were in agreement with those in previous research [30, 36, 37].
In this study, these histological alterations were accompanied by an insignificant difference in kidney weight, which was in line with the study by Denic et al. . They attributed these alterations to the combination of the age-related renal sinus fat increase and increased medullary volume with the decrease in cortical volume.
The histological changes in the aged kidney in this work were previously explained in different nephropathy models. The presence of periglomerular fibrosis, which disrupted glomerular outflow and led to cystic changes in Bowman's space, could explain the dilation of Bowman's space . On the other hand, glomerular hypercellularity, as a compensatory mechanism, could have resulted in the absence or narrowing of some Bowman's spaces .
Moreover, increased cell membrane permeability led to cytoplasmic vacuolation ; pyknotic nuclei indicated apoptotic changes . In addition, cast formation indicated tubular injury, as clarified by Rahman and Purwakanthi . Partial tubular obstruction or alterations in the structure of the tubular basement membrane were responsible for tubular dilatation . Furthermore, intense interstitial hemorrhage could be attributed to inflammation and vascular injury .
Glomerulosclerosis is considered one of the characteristic features of kidney aging . It may be attributed to many factors such as small renal arteriosclerosis with subsequent ischemic injury to nephrons , oxidative stress , and hypertension . In this study, glomerulosclerosis attributed to the presence of thick-walled blood vessels, increased systolic blood pressure, and elevation of oxidative markers.
In the current work, most histopathological changes were alleviated by exercise, which was in agreement with previous research examining distinct exercise modalities on different nephropathy models [49, 50]. On the contrary, Moningka et al.  reported that exercise did not effectively alter the age-induced chronic kidney changes in the aging Fisher 344 male rats. This discrepancy could be attributed to the use of different rat strain and exercise protocol.
The mechanisms by which treadmill exercise alleviated age-related kidney changes could be understood by investigating the pathophysiology of kidney aging. The pathogenesis of age-related kidney changes is still poorly clarified. Several clinical and experimental studies have shown that oxidative stress increases with normal aging, which was in line with our results [13, 52].
Moreover, this study proved that aging is associated with chronic low-grade systemic inflammation, agreeing with the results of a previous report . Renal fibrosis occurred due to elevated levels of inflammatory cytokine  with a subsequent imbalance between production and degradation of extracellular matrix, epithelial-to-mesenchymal transition, and fibroblast activation . In the present study, renal fibrosis was confirmed by the significantly increased percentage area of collagen deposition in the aged-S rats compared with that in young rats.
Furthermore, the favorable effects of exercise were attributed mostly to its antioxidant and anti-inflammatory nature. Both inactivity and high-intensity exercise increased oxidative stress, whereas moderate-intensity exercise reduces oxidative stress . Therefore, moderate-intensity exercise was performed in this work.
The antioxidant effect of exercise was confirmed in this study. According to a previous study , exercise training significantly improved the oxidant/antioxidant imbalance in the aged rats. Sallam and Laher  indicated that the anti-inflammatory effect of exercise is attributed to its role in the modulation of anti-inflammatory/proinflammatory cytokine profiles. The effect of exercise on different inflammatory markers is controversial. In this study, exercise training reduced plasma CRP but not IL-6 levels in aged-Ex rats compared with those of aged-S rats, which could be because CRP is more responsive to physical activity than IL-6 . Furthermore, in the present study, treadmill exercise alleviated the progression of renal fibrosis in the aged rats mostly due to its anti-inflammatory properties, which was in line with Huang et al. . They noted that renal fibrosis was reduced with exercise in a hypertensive rat model.
Different mechanisms are also implicated in renal pathology in aged-S rats. Hence, studying the modulation of these mechanisms reflected the beneficial role of treadmill exercise on the aged kidney. The present study revealed that podocyte injury, indicated by upregulation of desmin, and downregulation of VEGF, autophagy, and AQP3 were implicated in aging-induced renal changes, which was in line with Poulaki et al.  and Durvasula and Shankland , who attributed the different aging-related renal changes to podocyte injury.
Moreover, Yamaji et al.  observed a reduction in the VEGF gene in the aged kidney. Low VEGF could be responsible for podocyte injury, glomerulosclerosis, and tubulointerstitial fibrosis development . In addition, a low VEGF level is implicated in endothelial dysfunction with subsequent hypertension . Peralta et al.  declared a strong association between kidney function and systolic blood pressure.
Now, the expression and modulation of AQPs are intensely investigated around the world . In this study, the downregulation of AQP3 immunoreaction in aged-S rats was consistent with a previous report . Preisser et al.  studied AQP1-4 expression in the aged kidney. They reported no change in the expression of AQP1 and AQP4 and found downregulation of AQP2 and AQP3. Indeed, Lei et al.  confirmed that AQP3 deletion provoked kidney injury by increased apoptosis. Moreover, Xie et al.  attributed the reduction in AQP3 expression to increased oxidative stress.
The impact of autophagy on aging and progression of kidney disease is still not fully understood . Autophagy-related proteins are involved in the execution of autophagy. Beclin-1 participates in the early stages of autophagy. It promotes the nucleation of the autophagic vesicle and recruits proteins from the cytosol . In this study, autophagy was downregulated in the aged kidneys, which was in line with Cui et al. , who observed downregulation of autophagy-related gene light chain 3 in the aged kidneys. The precise mechanisms leading to age-associated autophagy reduction remain unclear, which could be due to a general failure in maintaining autophagy-related protein expression .
In the current study, treadmill exercise modulated different mechanisms via protecting the podocytes from injury, improving VEGF and AQP3 expression, and inducing kidney autophagy. Agreeing with these results, Ishikawa et al.  attributed the beneficial effects of exercise in diabetic kidney disease to the maintenance of podocytes, with alleviation of oxidative damage and inflammation.
Furthermore, the improvement in systolic blood pressure in aged-Ex rats was in line with previous report , which could be explained partially by upregulation in VEGF. Pourheydar et al.  considered exercise an innovative nonpharmacological therapy that adjusted VEGF protein level in the aged heart and hypothesized that exercise improved angiogenesis. The exercise-induced VEGF upregulation and the subsequent alleviation of the age-induced microvascular deterioration could be attributed to the antioxidant mechanism of exercise, as postulated by Viboolvorakul and Patumraj  due to the repeated exposure to increases in blood flow and shear stress during exercise .
Indeed, the significant increase in AQP3 immunoreactivity was consistent with the results of Amer and Al-Sharaky  who noted that swimming exercise modulated AQP3 renal expression. The exact mechanism by which exercise improves the AQP3 expression in the aged kidney is not well understood. However, the improvement of AQP3 expression could be attributed to the antioxidant properties of exercise since the impairment of AQP3 in the aged kidney was related to the aging-associated oxidative stress. Further studies are warranted to investigate in-depth the exact mechanism.
Moreover, Brandt et al.  stated that exercise could induce autophagy and considered exercise a stress stimulus, modulating cellular signaling and promoting metabolic adaptation. Zhou et al.  suggested that podocyte autophagy is involved in renal protection and may be a therapeutic target. Aerobic exercise could improve autophagy due to its capability of inhibiting the phosphorylation of mammalian target of rapamycin by upregulating the activity of adenosine monophosphate-activated protein kinase, thus ameliorating aging-associated changes. .
The present study demonstrates that moderate-intensity treadmill exercise training improved structural and functional age-related changes in the kidney of aged rats. Thus, aerobic exercise is highly recommended for elderly people for protection against kidney aging. Further studies are warranted to investigate in-depth the different underlying mechanisms.
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this article.
All the authors participated in the design, conducted the experiment, performed analysis and interpretation of the results, and wrote the manuscript.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
E. F. Melchioretto, M. Zeni, D. A. D. L. Veronez, E. L. Martins Filho, and R. D. Fraga, “Quantitative analysis of the renal aging in rats. Stereological study,” Acta Cirurgica Brasileira, vol. 31, no. 5, pp. 346–352, 2016.View at: Publisher Site | Google Scholar
J. R. Weinstein and S. Anderson, “The aging kidney: physiological changes,” Advances in Chronic Kidney Disease, vol. 17, no. 4, pp. 302–307, 2010.View at: Publisher Site | Google Scholar
K. Abdel-Kader and P. M. Palevsky, “Acute kidney injury in the elderly,” Clinics in Geriatric Medicine, vol. 25, no. 3, pp. 331–358, 2009.View at: Publisher Site | Google Scholar
K. Nitta, K. Okada, M. Yanai, and S. Takahashi, “Aging and chronic kidney disease,” Kidney & Blood Pressure Research, vol. 38, no. 1, pp. 109–120, 2013.View at: Publisher Site | Google Scholar
Y. Xie, B. Bowe, A. H. Mokdad et al., “Analysis of the Global Burden of Disease study highlights the global, regional, and national trends of chronic kidney disease epidemiology from 1990 to 2016,” Kidney International, vol. 94, no. 3, pp. 567–581, 2018.View at: Publisher Site | Google Scholar
R. J. Glassock and A. D. Rule, “The implications of anatomical and functional changes of the aging kidney: with an emphasis on the glomeruli,” Kidney International, vol. 82, no. 3, pp. 270–277, 2012.View at: Publisher Site | Google Scholar
E. D. O’Sullivan, J. Hughes, and D. A. Ferenbach, “Renal aging: causes and consequences,” Journal of the American Society of Nephrology, vol. 28, pp. 407–420, 2017.View at: Google Scholar
A. Denic, J. C. Lieske, H. A. Chakkera et al., “The substantial loss of nephrons in healthy human kidneys with aging,” Journal of the American Society of Nephrology, vol. 28, no. 1, pp. 313–320, 2017.View at: Publisher Site | Google Scholar
R. Schmitt and A. Melk, “Molecular mechanisms of renal aging,” Kidney International, vol. 92, no. 3, pp. 569–579, 2017.View at: Publisher Site | Google Scholar
R. Glassock and A. Rule, “The kidney in ageing: biology, anatomy, physiology and clincial relevance,” Oxford Textbook of Clinical Nephrology, Oxford University Press, Oxford, UK, 2016.View at: Google Scholar
H. Xie, F. Liu, L. Liu et al., “Protective role of AQP3 in UVA-induced NHSFs apoptosis via Bcl2 up-regulation,” Archives of Dermatological Research, vol. 305, no. 5, pp. 397–406, 2013.View at: Publisher Site | Google Scholar
J. He and B. Yang, “Aquaporins in renal diseases,” International Journal of Molecular Sciences, vol. 20, no. 2, p. 366, 2019.View at: Publisher Site | Google Scholar
H. Vlassara, M. Torreggiani, J. B. Post, F. Zheng, J. Uribarri, and G. E. Striker, “Role of oxidants/inflammation in declining renal function in chronic kidney disease and normal aging,” Kidney International, vol. 76, pp. S3–S11, 2009.View at: Publisher Site | Google Scholar
G. Gennaro, C. Ménard, S.-E. Michaud, and A. Rivard, “Age-Dependent impairment of reendothelialization after arterial injury,” Circulation, vol. 107, no. 2, pp. 230–233, 2003.View at: Publisher Site | Google Scholar
L. Wang, J. Wang, D. Cretoiu, G. Li, and J. Xiao, “Exercise-mediated regulation of autophagy in the cardiovascular system,” Journal of Sport and Health Science, vol. 9, no. 3, pp. 203–210, 2020.View at: Publisher Site | Google Scholar
O. Lenoir, P.-L. Tharaux, and T. B. Huber, “Autophagy in kidney disease and aging: lessons from rodent models,” Kidney International, vol. 90, no. 5, pp. 950–964, 2016.View at: Publisher Site | Google Scholar
T.-A. Lin, V. C.-C. Wu, and C.-Y. Wang, “Autophagy in chronic kidney diseases,” Cells, vol. 8, no. 1, p. 61, 2019.View at: Publisher Site | Google Scholar
Y. Kabeya, N. Mizushima, T. Ueno et al., “LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing,” The EMBO Journal, vol. 19, no. 21, pp. 5720–5728, 2000.View at: Publisher Site | Google Scholar
R. Kang, H. J. Zeh, M. T. Lotze, and D. Tang, “The Beclin 1 network regulates autophagy and apoptosis,” Cell Death & Differentiation, vol. 18, no. 4, pp. 571–580, 2011.View at: Publisher Site | Google Scholar
D. Ito, P. Cao, T. Kakihana et al., “Chronic running exercise alleviates early progression of nephropathy with upregulation of nitric oxide synthases and suppression of glycation in zucker diabetic rats,” PLoS One, vol. 10, no. 9, p. e0138037, 2015.View at: Publisher Site | Google Scholar
G. Hu, L. Xu, Y. Ma, M. Kohzuki, and O. Ito, “Chronic exercise provides renal-protective effects with upregulation of fatty acid oxidation in the kidney of high fructose-fed rats,” American Journal of Physiology - Renal Physiology, vol. 318, no. 3, pp. F826–F834, 2020.View at: Publisher Site | Google Scholar
F. Villanego, J. Naranjo, L. A. Vigara et al., “Impact of physical exercise in patients with chronic kidney disease: systematic review and meta-analysis,” Nefrologia, vol. 40, no. 3, pp. 237–252, 2020.View at: Publisher Site | Google Scholar
A. Asad, J. O. Burton, and D. S. March, “Exercise as a therapeutic option for acute kidney injury: mechanisms and considerations for the design of future clinical studies,” BMC Nephrology, vol. 21, pp. 446–511, 2020.View at: Publisher Site | Google Scholar
S. A. Rubin and D. Mickle, “A simply constructed treadmill for rodent exercise studies,” Journal of Applied Physiology, vol. 52, no. 2, pp. 505–507, 1982.View at: Publisher Site | Google Scholar
P. M. Sosa, B. S. Neves, G. S. Carrazoni et al., “Maternal deprivation induces memory deficits that are reduced by one aerobic exercise shot performed after the learning session,” Neural Plasticity, vol. 2019, Article ID 3608502, 11 pages, 2019.View at: Publisher Site | Google Scholar
T. Bazzano, T. I. Restel, L. C. Porfirio, A. S. D. Souza, and I. S. Silva, “Renal biomarkers of male and female Wistar rats (Rattus norvegicus) undergoing renal ischemia and reperfusion,” Acta Cirurgica Brasileira, vol. 30, no. 4, pp. 277–288, 2015.View at: Publisher Site | Google Scholar
M. Funamoto, H. Masumoto, K. Takaori et al., “Green tea polyphenol prevents diabetic rats from acute kidney injury after cardiopulmonary bypass,” The Annals of Thoracic Surgery, vol. 101, no. 4, pp. 1507–1513, 2016.View at: Publisher Site | Google Scholar
M. Schaier, I. Lehrke, K. Schade et al., “Isotretinoin alleviates renal damage in rat chronic glomerulonephritis,” Kidney International, vol. 60, no. 6, pp. 2222–2234, 2001.View at: Publisher Site | Google Scholar
N. Ebert, O. Jakob, J. Gaedeke et al., “Prevalence of reduced kidney function and albuminuria in older adults: the Berlin Initiative Study,” Nephrology Dialysis Transplantation: Official Publication of the European Dialysis and Transplant Association-European Renal Association, vol. 32, pp. 997–1005, 2017.View at: Publisher Site | Google Scholar
E. Costa, J. Fernandes, S. Ribeiro et al., “Aging is associated with impaired renal function, INF-gamma induced inflammation and with alterations in iron regulatory proteins gene expression,” Aging and disease, vol. 5, pp. 356–365, 2014.View at: Publisher Site | Google Scholar
C. G. Musso, J. Á. Gregori, J. R. Jauregui, and J. F. M. Núñez, “Creatinine, urea, uric acid, water and electrolytes renal handling in the healthy oldest old,” World Journal of Nephrology, vol. 1, no. 5, pp. 123–126, 2012.View at: Publisher Site | Google Scholar
V. R. Dharnidharka, C. Kwon, and G. Stevens, “Serum cystatin C is superior to serum creatinine as a marker of kidney function: a meta-analysis,” American Journal of Kidney Diseases, vol. 40, no. 2, pp. 221–226, 2002.View at: Publisher Site | Google Scholar
A. C. Baxmann, M. S. Ahmed, N. C. Marques et al., “Influence of muscle mass and physical activity on serum and urinary creatinine and serum cystatin C,” Clinical Journal of the American Society of Nephrology, vol. 3, no. 2, pp. 348–354, 2008.View at: Publisher Site | Google Scholar
E. B. Togoe, I. S. Silva, J. L. Cury, and F. A. Guarnier, “Muscle changes with high-intensity aerobic training in an animal model of renal disease,” Acta Cirurgica Brasileira, vol. 34, p. e201900503, 2019.View at: Publisher Site | Google Scholar
S. N. Hassan, W. Nargis, M. Khalil, M. Khalil, and M. R. Alam, “Corticomedullary index of human kidney,” Bangladesh Journal of Anatomy, vol. 10, pp. 20–22, 2012.View at: Google Scholar
J. H. Lim, E. N. Kim, M. Y. Kim et al., “Age-associated molecular changes in the kidney in aged mice,” Oxidative medicine and cellular longevity, vol. 2012, p. 171383, 2012.View at: Publisher Site | Google Scholar
A. Yabuki, S. Yoneshige, S. Tanaka, M. Tsujio, S. Mitani, and O. Yamato, “Age-related histological changes in kidneys of Brown Norway rat,” Journal of Veterinary Medical Science, vol. 76, 2013.View at: Google Scholar
A. Denic, R. J. Glassock, and A. D. Rule, “Structural and functional changes with the aging kidney,” Advances in Chronic Kidney Disease, vol. 23, no. 1, pp. 19–28, 2016.View at: Publisher Site | Google Scholar
W. Kriz, H. Hosser, B. Hahnel, N. Gretz, and A. Provoost, “From segmental glomerulosclerosis to total nephron degeneration and interstitial fibrosis: a histopathological study in rat models and human glomerulopathies,” Nephrology Dialysis Transplantation, vol. 13, no. 11, pp. 2781–2798, 1998.View at: Publisher Site | Google Scholar
A. ElMawla and H. Osman, “HPLC analysis and role of the Saudi Arabian propolis in improving the pathological changes of kidney treated with monosodium glutamate,” Spatula DD-Peer Reviewed Journal on Complementary Medicine and Drug Discovery, vol. 1, no. 3, pp. 119–127, 2011.View at: Publisher Site | Google Scholar
V. Filiopoulos and D. Vlassopoulos, “Inflammatory syndrome in chronic kidney disease: pathogenesis and influence on outcomes,” Inflammation and Allergy - Drug Targets, vol. 8, no. 5, pp. 369–382, 2009.View at: Publisher Site | Google Scholar
G. Kroemer, L. Galluzzi, P. Vandenabeele et al., “Classification of cell death: recommendations of the nomenclature committee on cell death 2009,” Cell Death & Differentiation, vol. 16, no. 1, pp. 3–11, 2009.View at: Publisher Site | Google Scholar
A. O. Rahman and A. Purwakanthi, “Kidney tubular injury of rats caused by unripe green betel nuts (Areca catechu),” Jurnal Kedokteran dan Kesehatan Indonesia, vol. 11, no. 1, pp. 27–33, 2020.View at: Publisher Site | Google Scholar
I. Sunila, “Cystic kidneys in copper exposed mussels,” Diseases of Aquatic Organisms, vol. 6, pp. 63–66, 1989.View at: Publisher Site | Google Scholar
K. S. Frazier, J. C. Seely, G. C. Hard et al., “Proliferative and nonproliferative lesions of the rat and mouse urinary system,” Toxicologic Pathology, vol. 40, no. 4_suppl, pp. 14S–86S, 2012.View at: Publisher Site | Google Scholar
W. K. Kremers, A. Denic, J. C. Lieske et al., “Distinguishing age-related from disease-related glomerulosclerosis on kidney biopsy: the Aging Kidney Anatomy study,” Nephrology Dialysis Transplantation, vol. 30, no. 12, pp. 2034–2039, 2015.View at: Publisher Site | Google Scholar
K. M. Rice, D. L. Preston, E. M. Walker, and E. R. Blough, “Aging influences multiple incidices of oxidative stress in the aortic media of the Fischer 344/NNia × Brown Norway/BiNia rat,” Free Radical Research, vol. 40, no. 2, pp. 185–197, 2006.View at: Publisher Site | Google Scholar
D. Duarte, C. Santos-Araújo, and A. F. Leite-Moreira, “Hypertension and angiogenesis in the aging kidney: a review,” Archives of Gerontology and Geriatrics, vol. 52, no. 3, pp. e93–e102, 2011.View at: Publisher Site | Google Scholar
G. Cao, J. González, A. Müller et al., “Beneficial effect of moderate exercise in kidney of rat after chronic consumption of cola drinks,” PLoS One, vol. 11, no. 3, p. e0152461, 2016.View at: Publisher Site | Google Scholar
B. H. Ali, T. Karaca, Y. Al Suleimani et al., “The effect of swimming exercise on adenine-induced kidney disease in rats, and the influence of curcumin or lisinopril thereon,” PloS one, vol. 12, no. 4, p. e0176316, 2017.View at: Publisher Site | Google Scholar
N. C. Moningka, A. L. Sindler, J. M. Muller-Delp, and C. Baylis, “Twelve weeks of treadmill exercise does not alter age-dependent chronic kidney disease in the Fisher 344 male rat,” The Journal of Physiology, vol. 589, no. 24, pp. 6129–6138, 2011.View at: Publisher Site | Google Scholar
S. Samarghandian, M. Azimi-Nezhad, A. Borji, and T. Farkhondeh, “Effect of crocin on aged rat kidney through inhibition of oxidative stress and proinflammatory state,” Phytotherapy Research, vol. 30, no. 8, pp. 1345–1353, 2016.View at: Publisher Site | Google Scholar
J. A. Woods, K. R. Wilund, S. A. Martin, and B. M. Kistler, “Exercise, inflammation and aging,” Aging and disease, vol. 3, pp. 130–140, 2012.View at: Google Scholar
A. Shankar, L. Sun, B. E. K. Klein et al., “Markers of inflammation predict the long-term risk of developing chronic kidney disease: a population-based cohort study,” Kidney International, vol. 80, no. 11, pp. 1231–1238, 2011.View at: Publisher Site | Google Scholar
G. Efstratiadis, M. Divani, E. Katsioulis, and G. Vergoulas, “Renal fibrosis,” Hippokratia, vol. 13, pp. 224–9, 2009.View at: Google Scholar
M. A. Bouzid, E. Filaire, A. McCall, and C. Fabre, “Radical oxygen species, exercise and aging: an update,” Sports Medicine, vol. 45, no. 9, pp. 1245–1261, 2015.View at: Publisher Site | Google Scholar
N. Okudan and M. Belviranli, “Effects of exercise training on hepatic oxidative stress and antioxidant status in aged rats,” Archives of Physiology and Biochemistry, vol. 122, no. 4, pp. 180–185, 2016.View at: Publisher Site | Google Scholar
N. Sallam and I. Laher, “Exercise modulates oxidative stress and inflammation in aging and cardiovascular diseases,” Oxidative medicine and cellular longevity, vol. 2016, p. 7239639, 2016.View at: Publisher Site | Google Scholar
C.-C. Huang, Y.-Y. Lin, A.-L. Yang, T.-W. Kuo, C.-H. Kuo, and S.-D. Lee, “Anti-renal fibrotic effect of exercise training in hypertension,” International Journal of Molecular Sciences, vol. 19, no. 2, p. 613, 2018.View at: Publisher Site | Google Scholar
E. Poulaki, M. G. Detsika, E. Fourtziala, E. A. Lianos, and H. Gakiopoulou, “Podocyte-targeted heme oxygenase (HO)-1 overexpression exacerbates age-related pathology in the rat kidney,” Scientific Reports, vol. 10, pp. 5719–5810, 2020.View at: Publisher Site | Google Scholar
R. V. Durvasula and S. J. Shankland, “Podocyte injury and targeting therapy: an update,” Current Opinion in Nephrology and Hypertension, vol. 15, no. 1, pp. 1–7, 2006.View at: Publisher Site | Google Scholar
M. Yamaji, H. Bielby, D. Licence et al., “VEGF-A loss in the haematopoietic and endothelial lineages exacerbates age-induced renal changes,” Microvascular Research, vol. 80, no. 3, pp. 372–383, 2010.View at: Publisher Site | Google Scholar
B. F. Schrijvers, A. Flyvbjerg, and A. S. De Vriese, “The role of vascular endothelial growth factor (VEGF) in renal pathophysiology,” Kidney International, vol. 65, no. 6, pp. 2003–2017, 2004.View at: Publisher Site | Google Scholar
S. R. Hayman, N. Leung, J. P. Grande, and V. D. Garovic, “VEGF inhibition, hypertension, and renal toxicity,” Current Oncology Reports, vol. 14, no. 4, pp. 285–294, 2012.View at: Publisher Site | Google Scholar
C. Peralta, M. Whooley, J. Ix, and M. Shlipak, “Kidney function and systolic blood pressure new insights from cystatin C: data from the Heart and Soul Study,” American Journal of Hypertension, vol. 19, no. 9, pp. 939–946, 2006.View at: Publisher Site | Google Scholar
G. Tamma, G. Valenti, E. Grossini et al., “Aquaporin membrane channels in oxidative stress, cell signaling, and aging: recent advances and research trends,” Oxidative medicine and cellular longevity, vol. 2018, Article ID 1501847, 14 pages, 2018.View at: Publisher Site | Google Scholar
G. Tamma, N. Goswami, J. Reichmuth, N. G. De Santo, and G. Valenti, “Aquaporins, vasopressin, and aging: current perspectives,” Endocrinology, vol. 156, no. 3, pp. 777–788, 2015.View at: Publisher Site | Google Scholar
L. Preisser, L. Teillet, S. Aliotti et al., “Downregulation of aquaporin-2 and -3 in aging kidney is independent of V2 vasopressin receptor,” American Journal of Physiology - Renal Physiology, vol. 279, no. 1, pp. F144–F152, 2000.View at: Publisher Site | Google Scholar
L. Lei, W. Wang, Y. Jia et al., “Aquaporin-3 deletion in mice results in renal collecting duct abnormalities and worsens ischemia-reperfusion injury,” Biochimica et Biophysica Acta - Molecular Basis of Disease, vol. 1863, no. 6, pp. 1231–1241, 2017.View at: Publisher Site | Google Scholar
B. Chifenti, M. T. Locci, G. Lazzeri et al., “Autophagy-related protein LC3 and Beclin-1 in the first trimester of pregnancy,” Clinical and experimental reproductive medicine, vol. 40, no. 1, p. 33, 2013.View at: Publisher Site | Google Scholar
J. Cui, X.-Y. Bai, S. Shi et al., “Age-related changes in the function of autophagy in rat kidneys,” Age, vol. 34, no. 2, pp. 329–339, 2012.View at: Publisher Site | Google Scholar
N. Martinez-Lopez, D. Athonvarangkul, and R. Singh, “Autophagy and aging,” Longevity Genes, vol. 847, pp. 73–87, 2015.View at: Publisher Site | Google Scholar
Y. Ishikawa, T. Gohda, M. Tanimoto et al., “Effect of exercise on kidney function, oxidative stress, and inflammation in type 2 diabetic KK-Ay mice,” Experimental Diabetes Research, vol. 2012, p. 702948, 2012.View at: Publisher Site | Google Scholar
J. Y. Wei, Y.-X. Li, and J. Ragland, “Effect of exercise training on resting blood pressure and heart rate in adult and aged rats,” Journal of Gerontology, vol. 42, no. 1, pp. 11–16, 1987.View at: Publisher Site | Google Scholar
B. Pourheydar, A. Biabanghard, R. Azari, N. Khalaji, and L. Chodari, “Exercise improves aging-related decreased angiogenesis through modulating VEGF-A, TSP-1 and p-NF-Ƙb protein levels in myocardiocytes,” Journal of Cardiovascular and Thoracic Research, vol. 12, no. 2, pp. 129–135, 2020.View at: Publisher Site | Google Scholar
S. Viboolvorakul and S. Patumraj, “Exercise training could improve age-related changes in cerebral blood flow and capillary vascularity through the upregulation of VEGF and eNOS,” BioMed Research International, vol. 2014, Article ID 230791, 12 pages, 2014.View at: Publisher Site | Google Scholar
C. S. Latimer, J. L. Searcy, M. T. Bridges et al., “Reversal of glial and neurovascular markers of unhealthy brain aging by exercise in middle-aged female mice,” PLoS One, vol. 6, no. 10, p. e26812, 2011.View at: Publisher Site | Google Scholar
R. Dalia, G. S. A. M. D. Al-Sharaky, and S. Ghada, “Modulation of renal aquaporin-3 by swimming exercise in fructose induced metabolic syndrome,” The Medical Journal of Cairo University, vol. 86, no. 12, pp. 4317–4325, 2018.View at: Publisher Site | Google Scholar
N. Brandt, T. P. Gunnarsson, J. Bangsbo, and H. Pilegaard, “Exercise and exercise training-induced increase in autophagy markers in human skeletal muscle,” Physiological reports, vol. 6, no. 7, p. e13651, 2018.View at: Publisher Site | Google Scholar
X.-J. Zhou, D. J. Klionsky, and H. Zhang, “Podocytes and autophagy: a potential therapeutic target in lupus nephritis,” Autophagy, vol. 15, no. 5, pp. 908–912, 2019.View at: Publisher Site | Google Scholar